During the past year, we have further refined the first in vitro bioassay system for qualitative and quantitative analysis of the testis stimulating factor. Until now, the only method for determining the presence of the testis stimulating factor utilized testicular artery and vein catheterization of intact cynomolgus monkeys. Using this bioassay, we have been able to show that this factor acts as a protein in ammonium sulfate precipitation. Using size exclusion chromatography, we can show two peaks of bioactivity which are distinct from LH. We have begun a collaboration with Dr. David Klapper at the University of North Carolina to help us with isolation of this factor. To date, we have used the standard isolation techniques of size, ion exchange, and immunoaffinity chromatography. We hope to have the factor isolated in the next year. In addition, we have further studied the second messenger system used by this factor. We will then develop antibodies to allow us to better measure it and to begin to look for the gene responsible for the factor. This factor may have unique clinical significance because it is able to stimulate virilization as well as spermatogenesis in the absence of gonadotropins. It may further be used as a model of a protein that can significantly affect reproduction and may further elucidate other pathways for reproductive effects of toxins.